Ketamine’s rapid antidepressant effects are mediated by Ca2+-permeable AMPA receptors

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    This paper addresses an important clinical concern which is how the antidepressant ketamine exerts its effects acts rapidly. The authors suggest the reason is that ketamine increases glutamatergic transmission in the hippocampus. The strengths are the data are mostly very good, and the limitations are a lack of compelling evidence that the hippocampus is the location where effects occur, as well as several other issues.

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Abstract

Ketamine is shown to enhance excitatory synaptic drive in multiple brain areas, which is presumed to underlie its rapid antidepressant effects. Moreover, ketamine’s therapeutic actions are likely mediated by enhancing neuronal Ca 2+ signaling. However, ketamine is a noncompetitive NMDA receptor (NMDAR) antagonist that reduces excitatory synaptic transmission and postsynaptic Ca 2+ signaling. Thus, it is a puzzling question how ketamine enhances glutamatergic and Ca 2+ activity in neurons to induce rapid antidepressant effects while blocking NMDARs in the hippocampus. Here, we find that ketamine treatment in cultured mouse hippocampal neurons significantly reduces Ca 2+ and calcineurin activity to elevate AMPA receptor (AMPAR) subunit GluA1 phosphorylation. This phosphorylation ultimately leads to the expression of Ca 2+ -Permeable, GluA2-lacking, and GluA1-containing AMPARs (CP-AMPARs). The ketamine-induced expression of CP-AMPARs enhances glutamatergic activity and glutamate receptor plasticity in cultured hippocampal neurons. Moreover, when a sub-anesthetic dose of ketamine is given to mice, it increases synaptic GluA1 levels, but not GluA2, and GluA1 phosphorylation in the hippocampus within 1 hr after treatment. These changes are likely mediated by ketamine-induced reduction of calcineurin activity in the hippocampus. Using the open field and tail suspension tests, we demonstrate that a low dose of ketamine rapidly reduces anxiety-like and depression-like behaviors in both male and female mice. However, when in vivo treatment of a CP-AMPAR antagonist abolishes the ketamine’s effects on animals’ behaviors. We thus discover that ketamine at the low dose promotes the expression of CP-AMPARs via reduction of calcineurin activity, which in turn enhances synaptic strength to induce rapid antidepressant actions.

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  1. Author Response

    Reviewer #1 (Public Review):

    This paper is based on the premise that ketamine exerts antidepressant effects that are rapid by increasing glutamatergic transmission. However, the authors note that how this effect occurs is unclear because ketamine antagonizes the NMDA receptor, a glutamatergic receptor. Others have suggested a compensatory change in the glutamatergic transmission and the authors suggest how this might occur. The authors should clarify if prior studies suggested a mechanism different from theirs and if so, which might be correct.

    There are also other mechanisms, such as the block of NMDA receptors on interneurons and the disinhibition of principal cells. It is important to clarify if this has already been addressed in the literature. Also, if their cultures are primarily glutamatergic neurons or they include interneurons and glia.

    The authors show calcineurin is reduced after ketamine exposure and this increases AMPA receptor GluA1 phosphorylation. They also show that Calcium permeable AMPA receptors (CP-AMPARs) increase.

    They also use suggest that the CP-AMPARs and other changes lead to enhanced synaptic plasticity, which could lead to antidepressant effects.

    Although a lot of work is done in cultured hippocampal neurons, 14 days in vitro, they show effects in vivo that are consistent with the data from cultures. For example, ketamine increases GluA1 phosphorylation. Also, blocking CPAMPARs in vivo reduces anxiety/depressive behaviors such as the open field and tail suspension tests.

    Overall the study appears to be done well and the presentation, writing, and references are good. There are important concerns regarding statistics, behavior, and pharmacology and several minor concerns.

    Major concerns

    1. Statistics.

    What was the stat test if the control was always 1? Often the control group is 1.00 with no SD but in other tests, the control group is 1.000 with an SD.

    In the previous submission, we neglected to include this information. Immunoblotting data have variable raw values; hence, the control group was used to normalize each group and was compared to the experimental groups. Thus, the control value for immunoblotting was always 1.000 without SD. Similarly, for imaging data, the average peak amplitude in control cells was used to normalize the peak amplitude in each cell and was compared to the experimental groups' average; thus, the control group is 1.000 with SD. The Franklin A. Graybill Statistical Laboratory at Colorado State University has been consulted for statistical analysis in the current study, including sample size determination, randomization, experiment conception and design, data analysis, and interpretation. Grouped results of single comparisons were tested for normality with the Shapiro-Wilk normality or Kolmogorov-Smirnov test and analyzed using the unpaired two-tailed Student’s t-test when data are normally distributed. Differences between multiple groups with normalized data were assessed by nonparametric Kruskal-Wallis test with the Dunn’s test.

    1. Behavior.

    It is not clear that the open field and tail suspension tests measure antidepressant actions. Why were more standard tests such as forced swim or sucrose preference, novelty-suppressed feeding, etc not used?

    We agree with the Reviewer’s concern. However, both the open field test and tail suspension test have long been used to determine animals’ anxiety-like and depression-like behaviors, respectively, in rodents (Seibenhener and Wooten, 2015; Ueno et al., 2022). Specifically, the open field test has been widely used to measure the ketamine effects on anxiety-like behavior in rodents (Guarraci et al., 2018; Pitsikas et al., 2019; Shin et al., 2019; Akillioglu and Karadepe, 2021; Yang et al., 2022; Acevedo et al., 2023). The tail suspension test has also been used to examine the ketamine effects on depression-like behavior in animals (Fukumoto et al., 2017; Yang et al., 2018; Ouyang et al., 2021; Rawat et al., 2022; Viktorov et al., 2022). Studies suggest that the forced swim test and the tail suspension test are based on the same principle: measurement of immobility duration while rodents are exposed to an inescapable situation (Castagne et al., 2011). Importantly, it has been suggested that the tail suspension test is more sensitive to antidepressant agents than the forced swim test because the animal will remain immobile longer in the tail suspension test than the forced swim test (Cryan et al., 2005). For this reason, we chose to use the tail suspension test instead of the forced swim test. This information has now been included in the revised manuscript. Additionally, because ketamine produces antidepressant effects within one hour after administration in humans (Berman et al., 2000; Zarate et al., 2006; Liebrenz et al., 2009), our study aims to understand the mechanism underlying ketamine's rapid (less than an hour) antidepressant effects. Given that sucrose preference test and the novelty suppressed feeding test need multiple days, it would not be suitable to achieve our goals.

    1. Pharmacology.

    The conclusions rest on the specificity of drugs.

    Is 5 uM FK506 specific?

    20 μM 1-naphthyl acetyl spermine (NASPM)?

    10 mg/kg IEM-1460?

    We neglected to add the rationale for the drug concentrations in the previous submission. Previous research, including our own, has employed FK506 at a variety of different concentrations to inhibit neuronal calcineurin activity (1 - 50 μM) (Hsieh et al., 2006; Schwartz et al., 2009; Kim and Ziff, 2014). Specifically, we have shown that 5 μM FK506 treatment for 12 hours significantly reduces neuronal calcineurin activity to increase GluA1 phosphorylation, which induces the expression of CP-AMPARs to elevate AMPAR-mediated synaptic activity (Kim and Ziff, 2014). Moreover, previous studies, including our own, have used NASPM at a variety of different concentrations to inhibit CP-AMPARs (3 - 250 μM) (Tsubokawa et al., 1995; Koike et al., 1997; Noh et al., 2005; Nilsen and England, 2007; Hou et al., 2008; Kim and Ziff, 2014). In fact, we have shown that 20 μM NASPM significantly reduces CP-AMPAR-mediated synaptic and Ca2+ activity (Kim and Ziff, 2014; Kim et al., 2015b). Finally, multiple reports demonstrate that 10 mg/kg IEM-1460 significantly reduces in vivo CP-AMPAR activity (Wiltgen et al., 2010; Szczurowska and Mares, 2015; Adotevi et al., 2020). This information has now been included in the revised manuscript.

    Reviewer #3 (Public Review):

    Ketamine has been shown to be effective at producing a rapid-antidepressant effect at low doses, but the underlying molecular mechanism of this effect is still not clear. Previous studies have suggested that the effect of low-dose ketamine may occur by promoting neuronal plasticity in the hippocampus. However, this goes against the findings that ketamine acts as a noncompetitive NMDA receptor antagonist, which should prevent NMDAR-dependent plasticity. Furthermore, a therapeutic dose of ketamine has been shown to increase neuronal Ca2+ signaling, which again does not conform to its antagonistic action on NMDA receptors. In this paper, the authors provide evidence that therapeutic low-dose ketamine increases the expression of Ca2+-permeable AMPA receptors (CP-AMPARs) by increasing phosphorylation of GluA1 subunit of AMPARs and surface expression of GluA1-containing CP-AMPARs. They further provide evidence that this is likely mediated by a decrease in calcineurin activity and that blocking CP-AMPARs prevent the antidepressant effect of ketamine in mice. One interesting finding of this study is that the authors see heightened sensitivity of ketamine in female mice, both at the level of behavioral readout and for molecular correlates. This finding is interesting in light of the different pharmacokinetics of ketamine reported in females and that ketamine metabolites can bind estrogen receptors.

    Based on their data and previous findings, the authors outline a plausible molecular signaling mechanism for the antidepressant effect of ketamine. Specifically, the authors propose that reduced neuronal activity, which could be triggered by ketamine-induced NMDAR antagonism, causes homeostatic plasticity to upregulate GluA1-containing CP-AMPARs. Their data would support this idea, as phosphorylation of GluA1 as well as increased surface expression and functional incorporation of CP-AMPARs at synapses have been shown before in models of homeostatic plasticity.

    1. Overall, the study is well-done and the data presented support the main conclusions. One main question is whether the current finding provides a conceptual advancement in our understanding of the molecular signaling involved in ketamine's antidepressant effects.

    We thank the reviewer's critique. In fact, research suggests multiple potential mechanisms of ketamine-induced neural plasticity. The main mechanism by which ketamine produce their therapeutic benefits on mood recovery is the enhancement of neural plasticity in the hippocampus (Miller et al., 2016; Aleksandrova et al., 2020; Kavalali and Monteggia, 2020; Grieco et al., 2022). However, ketamine is a noncompetitive NMDAR antagonist that inhibits excitatory synaptic transmission (Anis et al., 1983). A hypothesis to explain these paradoxical effects is that ketamine acts via direct inhibition of NMDARs localized on inhibitory interneurons, leading to disinhibition of excitatory neurons and a resultant rapid increase in glutamatergic synaptic activity to activate Ca2+ signaling pathway (Deyama and Duman, 2020; Gerhard et al., 2020). This stimulates the brain-derived neurotrophic factor (BDNF) signal pathway, which subsequently increases the translation and synthesis of synaptic proteins to enhance AMPAR-mediated synaptic plasticity (Deyama and Duman, 2020). Another potential explanation is that ketamine inhibits NMDARs on excitatory neurons, which induces a cell-autonomous form of homeostatic synaptic plasticity resulting in increased excitatory synaptic drive onto these neurons (Miller et al., 2016; Kavalali and Monteggia, 2020). Homeostatic synaptic plasticity is a negative-feedback response employed to compensate for functional disturbances in neurons and expressed via the regulation of AMPAR trafficking and synaptic expression (Wang et al., 2012). According to this hypothesis, ketamine disrupts basal activation of NMDARs on excitatory neurons, which engages a mechanism of homeostatic synaptic plasticity that results in a rapid compensatory increase in synaptic AMPAR expression in these neurons in a protein-synthesis dependent manner (Kavalali and Monteggia, 2023). Additionally, there is a NMDAR inhibition-independent mechanism mediated by hydroxynorketamine (HNK), the ketamine metabolite that lacks NMDAR inhibition properties (Carrier and Kabbaj, 2013; Franceschelli et al., 2015; Zanos et al., 2016). The current study offers a new neurobiological basis for ketamine’s actions that depend on the NMDAR inhibition-mediated elevation of GluA1-containing AMPAR trafficking, which is likely independent from the previous described mechanisms including the BDNF-induced protein synthesis-dependent (Deyama and Duman, 2020) or the NMDAR inhibition-independent pathway (Carrier and Kabbaj, 2013; Franceschelli et al., 2015; Zanos et al., 2016). Nonetheless, there are still many important questions surrounding the molecular mechanisms of ketamine's actions. This new information has now been included in the revised manuscript.

    1. There are previous studies that showed an increase in CP-AMPARs in the nucleus accumbens and an increase in the expression of GluA1 in the hippocampus with low-dose ketamine. In addition, ketamine's antidepressant effect has been shown to require GluA1 phosphorylation. The main contribution of this paper might be that it provides the potential molecular signaling within the same preparation (i.e. hippocampal neurons) and provides a causal link of CP-AMPARs in mediating the behaviorally measured antidepressant effect of ketamine.

    The study showing that ketamine induces the insertion of CP-AMPARs in the nucleus accumbens did not examine whether this change resulted in antidepressant behaviors (Skiteva et al., 2021). Therefore, it is difficult to conclude that the ketamine-induced expression of CP-AMPARs in the nucleus accumbens plays a role in behaviors. Moreover, as described above, a recent study shows that the hippocampus is selectively targeted by ketamine (Davoudian et al., 2023). We thus chose the hippocampus as our experimental model to test our hypothesis. However, we are unable to rule out the potential role of nucleus accumbens in ketamine’s antidepressant actions.

    1. Another question is whether the behavioral effect of ketamine is due to molecular changes in the hippocampus as outlined in this paper. A more targeted inhibition of CP-AMPAR function could resolve this issue. With the systemic application of CP-AMPAR antagonist as done in this study, it would be hard to know the role of CP-AMPAR upregulation in the hippocampus in mediating ketamine's effect. Especially, considering that low-dose ketamine has been shown to upregulate CP-AMPARs in the nucleus accumbens. While it would have been nice to know the site of action, this does not alter the conclusion that CP-AMPARs are involved in mediating the antidepressant effect of ketamine on behavioral readouts.

    We agree with this point. We have thus removed “the hippocampus” in the title and have further made equivalent revisions in the other parts of the revised manuscript.

  2. eLife assessment

    This paper addresses an important clinical concern which is how the antidepressant ketamine exerts its effects acts rapidly. The authors suggest the reason is that ketamine increases glutamatergic transmission in the hippocampus. The strengths are the data are mostly very good, and the limitations are a lack of compelling evidence that the hippocampus is the location where effects occur, as well as several other issues.

  3. Reviewer #1 (Public Review):

    This paper is based on the premise that ketamine exerts antidepressant effects that are rapid by increasing glutamatergic transmission. However, the authors note that how this effect occurs is unclear because ketamine antagonizes the NMDA receptor, a glutamatergic receptor.
    Others have suggested a compensatory change in the glutamatergic transmission and the authors suggest how this might occur. The authors should clarify if prior studies suggested a mechanism different from theirs and if so, which might be correct.

    There are also other mechanisms, such as the block of NMDA receptors on interneurons and the disinhibition of principal cells. It is important to clarify if this has already been addressed in the literature. Also, if their cultures are primarily glutamatergic neurons or they include interneurons and glia.

    The authors show calcineurin is reduced after ketamine exposure and this increases AMPA receptor GluA1 phosphorylation. They also show that Calcium permeable AMPA receptors )CP-AMPARs) increase.

    They also use suggest that the CP-AMPARs and other changes lead to enhanced synaptic plasticity, which could lead to antidepressant effects.

    Although a lot of work is done in cultured hippocampal neurons, 14 days in vitro, they show effects in vivo that are consistent with the data from cultures. For example, ketamine increases GluA1 phosphorylation. Also, blocking CPAMPARs in vivo reduces anxiety/depressive behaviors such as the open field and tail suspension tests.

    Overall the study appears to be done well and the presentation, writing, and references are good. There are important concerns regarding statistics, behavior, and pharmacology and several minor concerns.

    Major concerns
    1. Statistics.
    What was the stat test if the control was always 1?
    Often the control group is 1.00 with no SD but in other tests, the control group is 1.000 with an SD.
    e.g., line 145: "(CTRL) (CTRL, 1.000 and ketamine, 1.598 {plus minus} 0.543, p = 145 0.0039), but not GluA2 (CTRL, 1.000 and ketamine, 1.121 {plus minus} 0.464, p = 0.6498"

    Line 188:
    Here the control group has a SD:
    Line 188 CTRL, 1.000 {plus minus} 0.106 and ketamine, 0.942 {plus minus} 0.051, p = 0.0170

    2. Behavior.
    It is not clear that the open field and tail suspension tests measure antidepressant actions. Why were more standard tests such as forced swim or sucrose preference, novelty-suppressed feeding, etc not used?

    3. Pharmacology.
    The conclusions rest on the specificity of drugs.
    Is 5 uM FK506 specific?
    20 μM 1-naphthyl acetyl spermine (NASPM)?
    10 mg/kg IEM-1460?

  4. Reviewer #2 (Public Review):

    The abstract and introduction framework asserts that ketamine's enhancement of excitatory synaptic drive in the hippocampus is presumed to underlie its rapid antidepressant effects. This is not the only, and perhaps not the primary effect mechanism suggested by prior experiments, also strongly implicating disinhibitory effects in the prefrontal cortex as necessary and sufficient to mediate antidepressant effects. Nevertheless, it is valuable to seek mechanistic motifs that provide multiple paths for explaining the seemingly counterintuitive effects where NMDAR blocker enhances excitatory transmission. These need not be conserved across brain regions and cell classes. The primary result of this study demonstrates that 1 hr-long ketamine application to cultured cells reduces calcineurin and GCaMP activity to elevate AMPA receptor subunit GluA1 phosphorylation and enhance the expression of Ca2+-permeable, GluA2-lacking (CP-)AMPARs. These observations are then evaluated in vivo, where calcineurin shows a similar response to ketamine and CP-AMPAR antagonist-abolished ketamine effects on behavior in the open field and tail suspension tests. One significant uncertainty this study helps resolve is whether GluA2-containing AMPARs are removed from synapses or whether GluA2-lacking AMPARs are inserted following ketamine administration. GCaMP imaging, FRET and glutamate uncaging assays provide a strong complement to biochemistry and in vivo data. There are several significant technical and conceptual limitations in this work, which substantially limit the extent of conclusions that can be drawn at this point.

    1. The age of neurons in cell culture experiments was 14 days in vitro (DIV), representing developing cultures that are just starting to form synapses. How these effects carry over to more mature cultures or adult animals is unclear.

    2. Phosphorylation analyses, forming the foundation of this work, are carried out 1 hr after ketamine treatment. This is prior to the observed clinical effects of ketamine and this point should be acknowledged. Whether and how long this effect lasts remains to be examined. If the goal is to highlight the earliest likely effects of ketamine that should precede potential clinical effects, this should be acknowledged, and in that case, the onset of effects should be clarified. At this point, the temporal features remain undersampled, with a single time-point.

    3. A lower dose (50%) treatment was used to evaluate potential sex differences in ketamine effects, which is not sufficiently justified, except post hoc based on behavioral data. The discussion section does consider potential factors that can account for observed differences.

    4. The 1-hr timeline to behavioral testing is fast, relative to clinical effects on behavior as well as behavioral effects measured in most studies using mouse models.

    5. Tail suspension test is broadly acknowledged as an inadequate model of antidepressant effects.

    6. There is no evidence from the in vivo experiments that effects in the hippocampus are due to direct actions of ketamine, as those reported for the cell culture studies. Intraperitoneal injections cannot be used to localize primary effects in vivo to the hippocampus, which would require local delivery.

    7. If (MNI)-caged L-glutamate was used at 1 μM concentration, as stated in methods, this is considerably below typical concentrations reported in the literature.

  5. Reviewer #3 (Public Review):

    Ketamine has been shown to be effective at producing a rapid-antidepressant effect at low doses, but the underlying molecular mechanism of this effect is still not clear. Previous studies have suggested that the effect of low-dose ketamine may occur by promoting neuronal plasticity in the hippocampus. However, this goes against the findings that ketamine acts as a noncompetitive NMDA receptor antagonist, which should prevent NMDAR-dependent plasticity. Furthermore, a therapeutic dose of ketamine has been shown to increase neuronal Ca2+ signaling, which again does not conform to its antagonistic action on NMDA receptors. In this paper, the authors provide evidence that therapeutic low-dose ketamine increases the expression of Ca2+-permeable AMPA receptors (CP-AMPARs) by increasing phosphorylation of GluA1 subunit of AMPARs and surface expression of GluA1-containing CP-AMPARs. They further provide evidence that this is likely mediated by a decrease in calcineurin activity and that blocking CP-AMPARs prevent the antidepressant effect of ketamine in mice. One interesting finding of this study is that the authors see heightened sensitivity of ketamine in female mice, both at the level of behavioral readout and for molecular correlates. This finding is interesting in light of the different pharmacokinetics of ketamine reported in females and that ketamine metabolites can bind estrogen receptors.

    Based on their data and previous findings, the authors outline a plausible molecular signaling mechanism for the antidepressant effect of ketamine. Specifically, the authors propose that reduced neuronal activity, which could be triggered by ketamine-induced NMDAR antagonism, causes homeostatic plasticity to upregulate GluA1-containing CP-AMPARs. Their data would support this idea, as phosphorylation of GluA1 as well as increased surface expression and functional incorporation of CP-AMPARs at synapses have been shown before in models of homeostatic plasticity.

    Overall, the study is well-done and the data presented support the main conclusions. One main question is whether the current finding provides a conceptual advancement in our understanding of the molecular signaling involved in ketamine's antidepressant effects. There are previous studies that showed an increase in CP-AMPARs in the nucleus accumbens and an increase in the expression of GluA1 in the hippocampus with low-dose ketamine. In addition, ketamine's antidepressant effect has been shown to require GluA1 phosphorylation. The main contribution of this paper might be that it provides the potential molecular signaling within the same preparation (i.e. hippocampal neurons) and provides a causal link of CP-AMPARs in mediating the behaviorally measured antidepressant effect of ketamine.

    Another question is whether the behavioral effect of ketamine is due to molecular changes in the hippocampus as outlined in this paper. A more targeted inhibition of CP-AMPAR function could resolve this issue. With the systemic application of CP-AMPAR antagonist as done in this study, it would be hard to know the role of CP-AMPAR upregulation in the hippocampus in mediating ketamine's effect. Especially, considering that low-dose ketamine has been shown to upregulate CP-AMPARs in the nucleus accumbens. While it would have been nice to know the site of action, this does not alter the conclusion that CP-AMPARs are involved in mediating the antidepressant effect of ketamine on behavioral readouts.